The present invention relates to a gene construct, a pharmaceutical preparation and their use.
The beginning of the era of gene therapy in medicine has been marked by the successful gene transfer of the adenosine desaminase gene to a child with severe immunodeficiency. To date findings coming mainly from animal experiments indicate that this form of therapy is not only useful in the correction of genetically caused diseases but also in the therapy of malignant neoplasias (Culver and Blaese, 1994).
The methods up to now being in the test phase are aimed at either a direct destruction or at least xe2x80x9cnormalizationxe2x80x9d of the tumor cell, or at the activation of an immune reaction directed against the tumor. The destruction by the transfer of so-called suicide genes or the normalization by the transfer of tumor suppressor genes requires the gene transfer being performed with high efficiency. The direct intratumour transfer of murine cell lines producing retroviruses containing the suicide gene thymidine kinase of herpes virus has been already performed in the case of multiform glioblastoma (Culver and Van Gilder, 1994).
As long as there is no efficient system available to achieve targeted gene transfer to all tumor cells in vivo, approaches involving the immune system in tracking down and destroying all tumor cells seem the most promising. However, a prerequisite of these approaches is the fundamental capability of the immune system to recognize the tumor cells by means of tumor-specific antigens, which appear on tumor cells and not on normal cells (Boon et al., 1994). For example, these tumor-specific antigens include viral gene products (e. g. gene products of the human papilloma virus in genital tumors) or mutationally altered oncogene products (e. g. the ras gene product or the tumor-specific bcr-abl fusion protein). Further suitable candidates for tumor specific antigens are the so-called idiotypes, i. e. immunoglobulins or T cell receptors on the cellular surfaces of B or T cell derived tumors. Recently, the identification of a plurality of tumor-associated antigens has been carried out, for example in malignant melanoma. These genes, however, are not exclusively expressed by the tumor, but to a small extent also by other somatic cells, such as melanocytes. The knowledge of tumor-specific or tumor-associated antigens, respectivly, is probably about to increase sharply because of the recent successful recovery and analysis by biochemical methods of the peptides presented by a tumor MHC complex (Mandelboim et al.; Cox et al., 1994).
Already in the mid-eighties, using an approach employing experiments on laboratory animals tumor cells were observed to loose their tumorigenicity in the syngeneic animal if the tumor cells were transfected with a cytokine expression vector (e. g. IL-2) by. gene transfer (Pardoll, 1993). This effect has also been observed in the case of a mixture of modified (i.e., cells that contained the expression vector) and non-modified cells. The local production of an immunostimulatory cytokine in a subset of the tumor cells is obviously capable of causing an immune reaction directed against the wild-type tumor. One of the most extensive studies of this kind using the murine malignant melanoma model B-16 showed retroviruses transducing GM-CSF, IL-4, and IL-6 to be most effective (Dranoff et al., 1993). The outcome of these observations was that a number of clinical studies on the subject of intratumour transfer of cytokines have been entered or are presently being entered worldwide (Foa et al., 1994). Generally, the protocols entail an ex vivo gene transfer into tumor cells which have been established in vitro for a short time period. In most of the protocols amphotrophic retroviruses are employed as vectors. After viral gene transfer, the cells are reimplanted into the patients. They can be irradiated prior to reimplantation. These approaches are, however, strongly limited by the technical difficulties of culturing the tumor cells in vitro even for a short time period. Therefore, a modification of this approach entails transducing or transfecting, respectively, either tumor infiltrating lymphocytes (TIL) (Treisman et al., 1995) or autologous fibroblast cells instead of the tumor cells themselves.
A further approach also referring to observations obtained from animal experiments in the eighties has been developed by Gary Nabel and already converted into a clinical protocol (Nabel et al., 1993; Plautz et al., 1993). This approach assumes that artificial allogenization of a subset of the tumor cells by the transfer of transplantation antigens may be sufficient to induce an immune reaction of the organism against the unmodified tumor cell. This protocol entails the direct transfer of an HLA B7 expression construct into the tumor using liposomes. Repeated injection of the HLA B7 gene construct into skin metastases of a moribund patient brought about regression of another untreated metastasis and of a pulmonary mestastasis, respectively (Nabel et al., 1993).
Many in vivo tumor cells lack the B7 surface antigen mediating co-stimulatory signals for T cell recognition. Therefore, attempts are made to stimulate the production of this signalling molecule in tumor cells by gene transfer (June et al., 1994; Li et al., 1994).
The aforementioned approaches to solve the problems bear the following disadvantages:
An advantage of amphotropic retroviruses is that integration of the proviral DNA into the target cell and the viral promoter/-enhancer combination generally permit a stable expression level during several cell divisions. The essential step of integration, however, bears the danger of insertional mutagenesis. Moreover, so-called xe2x80x9cpackagingxe2x80x9d cell lines produce relatively small amounts of recombinant virus which to date fail to be enriched because of their lability. Therefore, direct intratumour gene transfer is only possible using virus producing cells. Release of infectious virus in the target organism, however, may lead to infection of other dividing cells, such as intestinal epithelium or hematopoietic stem cells, after hematogenous transmission.
The liposome-mediated direct incorporation of DNA has been demonstrated successfully using the endothelium of large blood vessels (Ohno et al., 1994). An advantage of this approach is that it lacks the risk of insertional mutagenesis as well as of the undesirable remote effect; but this approach achieves only transient expression of the incorporated gene construct in dividing tissue because the DNA generally fails to be integrated or replicated.
One of the main technical obstacles is the in vitro culturing of tumor cells of every single patient. The performance of gene transfer into tumor cells cultured for a relatively short time period requires extraordinary experimental skills and is successful only in a portion of the cases. To date, infection with recombinant retroviruses represents the technique of choice for a gene transfer into this kind of cells.
EBV is present in lymphoblastoid cell lines (LCLs) in a state of latency. That means only a very small percentage of the infected cells produces infectious virus. In the state of latency, only six nuclear localized proteins (EBNA1, 2, 3A, B, C, LP) and two membrane-bound proteins (LMP and TP) of the virus are expressed. Generally, the EBV genome is present in the infected cell in episomal form in 10 to 100 copies. In the state of latency, the replication of the viral genome starts at an origin of replication (orip) (Yates et al., 1984). Maintenance of the episomal replication further requires binding of the EBNAl protein to the oriP (Yates et al., 1984). The EBV-derived vectors consist of pBR sequences, oriP, an EBNA-1 expression cassette, and a selection marker specific for eukaryotic cells (e. g. the hygromycin resistance gene). Furthermore, these vectors have the capacity for additional 20 to 30 kb of foreign sequences. Constructs based on these vectors (i) show very much better xe2x80x9cretentionxe2x80x9d in the cell even in the absence of selection (Middleton and Sugden, 1994); (ii) allow controlled expression without positional effects, and (iii) bear a substantially decreased risk of insertional mutagenesis. The efficiency of these vectors compared to mere plasmid vectors in obtaining stably transfected cells is substantially higher.
More than 95% of adult humans are infected by EBV. The primary infection occurs either asymptomatically or in the form of an infectious mononucleosis. The immunological control of the virus-infected cells in vivo has been very well investigated. The various latent gene products of the virus are recognized by specific cytotoxic T cells. Only in the state of extreme immunosuppression, e. g. as observed with AIDS patients or iatrogenically induced in transplant recipients, is there the possibility of polyclonally proliferating EBV-positive cells.
The synergistic way of function of the three regulatory elements (xcexaMAR, xcexaEi, xcexaE3xe2x80x2) of the immunoglobulin xcexa locus has first been demonstrated in the case of activation of the c-myc promoters P1 and P2 (Polack et al., 1993; Hxc3x6rtnagel et al., 1995). In a chromosomal translocation observed in Burkitt lymphoma (BL) a co-localisation of the c-myc gene and the region of the human immunoglobulin xcexa locus located 3xe2x80x2 of the xe2x80x9cxcexa joining regionxe2x80x9d occurs, by which the c-myc gene is activated. A characteristic of this activation is an alteration in the usage of the promoters of the c-myc gene: the P1 promoter is used preferentially over the P2 promoter. In contrast, in normal non-transformed cells the P2 promoter is used preferentially. Furthermore, derepression of the c-myc gene on the level of transcriptional elongation is observed.
To study the mechanism of c-myc activation by t(2;8) translocation the interaction of the c-myc gene with several regions of the Igxcexa locus was examined. As a technique the stable infection of BL cells by episomally replicating EBV derived vectors has been selected. Into these vectors the c-myc gene was cloned under the control of two regions of the IgK locus. One of these regions extends from the J region up to about 1,2 kb 3xe2x80x2 from the constant region (Cxcexa) while the second region encompasses the xcexa3xe2x80x2E located 12 kb 3xe2x80x2 of Cxcexa. Measurement of the c-myc expression obtained by this construct and by several shortened forms (deletions) resulted first in the observation that these regions are necessary and sufficient for the BL-specific activation of the c-myc gene (Polack et al., 1993). By further deletions the responsibility of three elements of the Igxcexa locus, xcexaMAR, xcexaEi, and xcexaE3xe2x80x2, for the activation of the c-myc gene could be demonstrated (Hxc3x6rtnagel et al, 1995). Furthermore, the chromatin structure of the contructs stably introduced into BL cells was examined by DNaseI mapping of hypersensitive sites (HSS). The typical HSSs were formed in the 5xe2x80x2 region of the c-myc gene as well as in portions of the Igxcexa locus. This demonstrates the formation of a normal chromatin structure on the extra-chromosomally replicating constructs.
Utilization of the Igxcexa elements in association with an episomal vector shows:
a synergistic activation of a heterologous promoter (c-myc) by the Igxcexa elements;
a large capacity of the episomal vectors;
the formation of a regular chromatin structure.
The constructs described in the publication by Hxc3x6rtnagel et al., 1995, were deletion constructs for the determination of regions of Ig which are essentially necessary for c-myc activation. It does not suggest the gene constructs according to the present invention containing the elements in a functional arrangement, and the use of the gene constructs according to the present invention.
One problem of the present invention to provide a gene construct for gene therapy avoiding the aforementioned disadvantages known from the state of the art.
This aim is achieved according to the invention by a gene construct containing, in functional association, at least:
(a)
(i) a combination of two enhancer elements of the immunoglobulin xcexa locus, namely the xcexa intron enhancer (xcexaEi) and the xcexa 3xe2x80x2 enhancer (xcexaE3xe2x80x2); or
(ii) a combination of two enhancer elements of the immunoglobulin heavy chain xcexc locus, namely xcexcEi and the xcexcE3xe2x80x2 enhancer region located 3xe2x80x2 of Cxcex1; or
(iii) a combination of one or more of these enhancer elements of (ii) together with one or more of the aforementioned elements of the immunoglobulin xcexa locus; or
(iv) the single enhancer element of the immunoglobulin xcex locus; or
(v) a combination of this enhancer element of (iv) together with one or more of the aforementioned elements of the immunoglobulin xcexa locus; or
(vi) a combination of this enhancer element of (iv) together with one or more of the above elements of the immunoglobulin heavy chain xcexc locus; and further
(b) a promoter;
(c) a gene of interest, selected from at least one element of the group, consisting of cytokine gene, viral antigen, cellular adhesion gene, tumor antigen, co-stimulatory signalling molecule gene, a HLA gene non-identical with the recipient, and xcex2-galactosidase gene; and
(d) a polyadenylation site (PAA).
Preferably, the gene construct according to the present invention contains the following combinations of regulatory elements:
xcexaEi and xcexE, xcexcEi and xcexaE3xe2x80x2, xcexcEi and xcexcE3xe2x80x2, xcexaEi and xcexcE3xe2x80x2, xcexcEi and xcexE, xcexaMAR and xcexaEi and xcexaE3xe2x80x2, xcexE and xcexaE3xe2x80x2, xcexE and xcexcE3xe2x80x2.
In a further preferred embodiment the gene construct of the present invention contains the immunoglobulin xcexa matrix attachment region (xcexaMAR) or the immunoglobulin xcexc matrix attachment region (xcexcMAR) in addition to one or more of the aforementioned enhancer elements or the combinations of enhancer elements.
The gene construct of the present invention is useful for gene therapy of diseases of the B cell system.
Preferably, the gene of interest may be the B7-1 or the B7-2 gene.
The gene construct of the present invention preferably contains sequences derived from EBV vectors, mini EBV vectors, bacterial vectors, from retroviruses, from adenovirus-associated viruses, from adenoviruses, or from vaccinia viruses. Further, it preferably contains sequences derived from bacterial vectors. In one embodiment of the invention, the sequence derived from EBV vectors or mini EBV vectors is the origin of replication (oriP).
In a preferred embodiment, the gene construct of the present invention additionally contains the EBNA1 expression cassette. Further, the gene construct of the present invention may additionally contain a marker gene, preferably being a resistance gene. This resistance gene is selected from resistance genes known as such. For example, an ampicillin resistance gene or a hygromycin resistance gene or a neomycin resistance gene may be employed.
The bacterial vector sequence may be chosen from any vector sequence known as such. Preferably, it is derived from pBR vectors.
In a particularly preferred embodiment, the gene construct of the present invention comprises EBV-derived vector sequences with or without the EBNA1 gene. Reference is made here to the complete contents of the publication of Sugden et al., 1985, and it shall be incorporated into this application for the completeness of the disclosure.
The promoter may be selected from any promoter capable of expressing the gene of interest in a selected cell. Preferably, the promoters of the group of a cell specific promoters, cytomegalovirus (CMV) promoter, tk and xcex2 globin promoters may be used.
The polyadenylation site may be derived from those sequences generally employed as a polyadenylation site. A representative polyadenylation site is the human xcex2 globin gene polyadenylation site or the SV 40 polyoma virus polyadenylation site.
The gene construct of the present invention allows the expression of a gene including therapeutically useful genes in a host cell. The protein encoded by the gene can then be produced by culturing the host cell under conditions that allow expression of the gene. Preferably, these therapeutically useful genes are expressed in B cells, B cell-derived cells, such as B cell tumor cells or cells immortalized by EBV or mini EBV, following successful gene transfer for the purpose of therapy of malignant and viral diseases.
Examples of the genes which may be expressed by the gene construct according to the invention are the cytokine genes, selected from the group of IL-2, -4, -6, -7, -8, -10, GM-CSF, G-CSF, TNF alpha, MCP 1, interferon gamma.
In a further embodiment of the present invention, into the construct of the present invention are inserted as a gene of interest a tumor antigen selected from the group of antigens members of human papilloma virus (HPV), melanoma-associated antigens, of the MAGE, BAGE, and GAGE gene family, the gp100, the idiotypes of T cell or B cell receptors of tumors, and mutated oncogenes. Particularly, the tumor antigens may be tumor-associated or tumor-specific antigens.
Preferably, the aforementioned tumor antigens are mutated oncogenes, such as ras genes or p53 genes or the derivatives thereof. The antigen associated with malignant melanoma is preferably the tyrosinase gene. Further genes of interest useful in the present invention are well-known to one skilled in the art and may be selected dependent on the disease to be treated.
The present invention also comprises such prokaryotic cells and eukaryotic cells transfected by one of the gene constructs of the present invention so as to contain the gene construct in an integrated or episomal, i. e. non-integrated, form. Preferably, the prokaryotic cell employed is an E. coli cell, and the eukaryotic cell employed is a B cell immortalized by EBV or mini EBV.
Preferably, the gene construct according to the present invention may contain as a gene of interest the viral antigens of HIV, CMV, HTLV1, and HPV, wherein said antigens are capable of inducing an immune reaction against the virus-infected cell.
The gene constructs according to the -invention may be used in the form of a pharmaceutical preparation further containing conventional carriers and/or excipients well known as such.
Preferably, the gene constructs of the invention are present in the pharmaceutical preparation according to the invention in liposomes or liposome-like structures.
In a further embodiment of the invention, the gene construct of the invention lacks a c-myc tumor antigen.
In a preferred embodiment of the invention there is provided a pharmaceutical preparation containing the gene construct of the invention in an effective amount together with conventional carriers and/or excipients.
In a further preferred embodiment of the present invention there is provided a pharmaceutical preparation containing primary B lymphocytes or fibroblast cells immortalized by EBV or mini EBV and comprising the gene construct of the present invention in an effective amount together with conventional carriers and/or excipients.
In a further preferred embodiment there is provided a pharmaceutical preparation containing primary B lymphocytes or fibroblast cells immortalized by EBV or mini 22V and comprising the gene construct of the present invention together with autologous T cells in an effective amount and conventional carriers and/or excipients.
In a further preferred embodiment there is provided a pharmaceutical preparation containing autologous T cells stimulated and expanded ex vivo using primary B lymphocytes or fibroblasts immortalized by EBV-or mini EBV and comprising the gene construct according to the invention.
By using an EBV-derived vector in combination with three specific regulatory elements of the immunoglobulin kappa locus, namely the matrix attachment region, the intron enhancer, and the 3xe2x80x2 enhancer, a therapeutically beneficial gene can be specifically expressed in B cells over a prolonged time period. A further advantage of the use of the gene constructs of the invention is the ommission of the culturing of primary B cells, which is very complicated experimentally since immortalized cells (LCL cells) of almost any individual can be obtained by infection of peripheral B cells with EBV or mini EBV (see review n: Rogers et al., 1992). The establishment of these LCLs is much less complicated experimentally than the culture of primary tumor cells.